Liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC/ESI-MSMS) has emerged as a powerful method to detect oxygenated derivatives of fatty acids including eicosanoids. With these methods it is possible to analyze a large collection of eicosanoids in a single LC/ESI-MSMS run. These lipid mediators are detected by multiple reaction monitoring in which parent ions are isolated in the first stage of the mass spectrometer followed by collision-induced dissociation to give fragment ions, which are detected after an additional stage of mass spectrometer isolation. The current limit of quantification of analytes is in the 10-20 pg range. This sensitivity level is appropriate for studies with cultured cells in vitro or with relatively large tissue samples, but it is not sufficient for studies with smaller volume samples such as joint synovial fluid or bronchoalveolar lavage fluid from experimental rodents.
For reasons that are not well understood, cations generally form gaseous ions better than anions in the electrospray ionization source of the mass spectrometer. Additionally, for underivatized carboxylic acids it is required to add a weak organic acid to the chromatographic mobile phase (e.g., formic acid) so that the carboxylic acid is kept in its protonated state, which allows it to be retained on the reverse-phase column to ensure chromatographic separation. However, the presence of the weak acid offsets the formation of carboxylate anions in the electrospray source because the weak acid carries most of the anionic charge in the electrospray droplets, and thus formation of analyte anions is suppressed.
However, despite the attractiveness of converting the carboxylic acid to a fixed charge cationic derivative, organic cations tend to fragment by collision-induced dissociation near the cationic site. Fragmentation in the derivatization tag is not desirable because analytes that form isobaric parent ions and that co-migrate on the LC column will not be distinguished in the mass spectrometer if they give rise to the same detected fragment ion. This loss of analytical specificity is a serious problem when analyzing complex biological samples. Fragmentation in the analyte portion rather than in the tag portion also reduces chemical noise, which also enhances sensitivity of detection.
Given the importance of oxygenated fatty acid derivatives in numerous medically important processes such as inflammation and resolution of inflammation, a need exists to improve the LC/ESI-MSMS sensitivity of detection of these lipid mediators using a widely available analytical platform. The present invention seeks to fulfill this need and provide further related advantages.